To study how Brd4 recognizes acetylated chromatin in vivo, we have introduced synthetic histone H4 tail peptides into tissue culture cells. The synthetic peptides were acetylated at various lysine residues that mimic the modification patterns of endogenous H4, and were fused to a peptide representing HIV-Tat. Due to the cell membrane penetrating activity of HIV-Tat, these peptides were efficiently taken up by the cells. We found that acetylated H4 peptides, but not unacetylated counterpart accelerated the recovery of Brd4 after photobleaching in live cells, indicating that acetylated H4 tail peptides bind to endogenous Brd4 and altered its interaction with the native chromatin. In support of the importance of Brd4-chromaitn interactions, acetylated peptides stimulated the release of Brd4 from mitotic chromosomes, resulting in the inhibition of mitotic progression. To address the role of Brd4 in mitotic gene expression, we studied binding of Brd4 to mitotic chromosomes. Chromatin immunoprecipitation analysis found that Brd4 preferentially binds to the transcription start sites of genes that are expressed immediately after mitosis. These regions also showed high levels of acetylation both in histone H3 and H4, indicating that Brd4 marks mitotic chromatin by recognizing acetylated histones and directs post-mitotic gene expression.